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KRAS mutations occur in 30-40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apc(flox/flox); LSL-Kras(G12D) and CDX2P-G22Cre;Apc(flox/flox) mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT-PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin-nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin-NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC.
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Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.
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Matriz Extracelular/metabolismo , Mutação , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/etiologia , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Animais , Crizotinibe , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/fisiologiaRESUMO
The transcribed-ultraconserved regions (T-UCRs) are a novel class of non-coding RNAs, which are absolutely conserved (100%) between the orthologous regions of the human, rat and mouse genomes. Previous studies have described that several T-UCRs show differential expressions in cancers and might be involved in cancer development. We investigated the transcriptional levels of representative 26 T-UCRs and determined the regions that were differently expressed in prostate cancer (PCa) and gastric cancer (GC). A quantitative reverse transcription-polymerase chain reaction analysis revealed the downregulation of Uc.158+A expression by a DNA methylation-associated mechanism, which was restored by 5-Aza-dC (5-aza-2'-deoxycytidine) treatment. Bisulfite genomic sequencing using cell lines and tissue samples demonstrated cancer-specific CpG hypermethylation in both GC and PCa. However, Uc.416+A was only overexpressed in GC and we identified an miR-153 binding site in the possible regulatory region of Uc.416+A using online databases. Along with a forced expression or knockdown of miR-153 in MKN-74 GC cells, the transcriptional levels of Uc.416+A were significantly disturbed. A luciferase reporter gene assay supported the direct regulation of Uc.416+A expression by miR-153. Furthermore, Uc.416+A was associated with cell growth through the regulation of IGFBP6 (insulin-like growth factor-binding protein 6) in GC. These findings suggest an oncogenic role of Uc.416+A in GC, which suggests that our approach would provide new insights into functional studies of T-UCRs in cancer biology.
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Metilação de DNA , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias da Próstata/genética , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Sequência Conservada/genética , DNA de Neoplasias/genética , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/patologiaRESUMO
We built an in-house oligonucleotide array on which 394 genes were selected based on our Serial Analysis of Gene Expression (SAGE) data and previously reported array data and listed several genes related to cancer progression. Among these, we focused on SEC11A, which encodes the SPC18 protein. SEC11A mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in gastric cancer (GC) tissue samples. Expression and distribution of SPC18 protein were investigated by immunohistochemical analysis in two independent GC cohorts (Hiroshima cohort, n=99 and Chiba cohort, n=989). To determine the effect of SPC18 on cell viability and invasiveness in vitro, MTT and Boyden chamber invasion assays were performed. To evaluate the influence of SPC18 on cell growth in vivo, GC cells were injected into severe combined immunodeficiency mice. Levels of TGF-α and EGF in media from the GC cells were measured by enzyme-linked immunosorbent assay (ELISA). Studies in human tissue revealed overexpression of SEC11A mRNA in 40% of 42 GC samples by qRT-PCR. Immunohistochemical analysis of SPC18 revealed that 26 and 20% of GC cases were SPC18-positive in the Hiroshima and Chiba cohorts, respectively. In both cohorts, the Kaplan-Meier analysis showed poorer survival in SPC18-positive GC cases than in SPC18-negative GC cases. Forced expression of SPC18 activates GC cell growth in vitro and in vivo. The levels of TGF-α in culture media from GC cells were reduced by knockdown of SPC18. These results indicate that SPC18 contributes to malignant progression through promotion of TGF-α secretion in GC.
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Peptídeo Hidrolases/metabolismo , Neoplasias Gástricas/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Idoso , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos SCID , Análise Multivariada , Transplante de Neoplasias , Peptídeo Hidrolases/genética , Modelos de Riscos Proporcionais , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Transcriptoma , Carga TumoralRESUMO
Gastric carcinogenesis is a multistep process, during which numerous genetic and epigenetic alterations accumulate: there are abnormalities of growth factors/receptors, angiogenic factors, cell cycle regulators, DNA mismatch repair genes etc. These abnormalities define, at the same time, the biological character of the cancer cell and may thus serve as therapeutic targets. Genetic instability may cause accumulation of genetic abnormalities. The most important epigenetic alterations are DNA methylation, histone modification and chromatin remodeling. Some of these changes are common in gastric cancer, regardless of subtype, and some differ by histological type or (gastric or intestinal) mucin phenotype. Genetic polymorphism is a crucial endogenous cause and fundamental aspect of cancer risk. Importantly, genetic polymorphisms are also associated with the therapeutic efficacy and toxicity of anti-cancer drugs. Genomic science and technology such as Serial Analysis of Gene Expression (SAGE) allows the identification of novel genes and molecules specifically up-regulated or down-regulated in gastric cancer, e.g., RegIV and claudin-18 can be identified. Advances in our understanding of the genetic and molecular bases lead to improved diagnosis, personalised medicine and prevention of gastric cancer.
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Neoplasias Gástricas/genética , Proteínas de Ciclo Celular/genética , Epigênese Genética , Expressão Gênica , Instabilidade Genômica , Infecções por Helicobacter , Helicobacter pylori/patogenicidade , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Polimorfismo Genético , Fatores de Risco , Neoplasias Gástricas/microbiologiaRESUMO
BRG1, a component of the SWI/SNF complex, regulates gene transcription through chromatin remodeling. Certain human cancer cell lines have been shown to contain homozygous deletions or mutations, half of which are concentrated in exons 4 and 10, resulting in aberrant BRG1 expression. We examined the expression of BRG1 in 38 gastric carcinomas and corresponding nonneoplastic mucosa by using the quantitative real-time RT-PCR method. Twenty-three carcinomas (61%) showed increased BRG1 expression in tumor tissue in comparison with that in nonneoplastic mucosa. The T/N ratio (the expression level of BRG1 mRNA in tumor tissues relative to those in corresponding nonneoplastic mucosa) in advanced cases of gastric carcinoma (stages III and IV) was significantly higher than that in cases of stage I and II carcinoma (p = 0.029). Furthermore, gastric carcinomas with lymph node metastasis showed a tendency to express BRG1 at a higher level than gastric carcinomas without metastasis (p = 0.097). We also searched for genetic alterations of the BRG1 gene in 8 gastric carcinoma cell lines and 33 primary gastric carcinomas by PCR-SSCP analysis. No SSCP variants in exons 4, 10 and 16 of the BRG1 gene were found in both gastric carcinoma cell lines and primary gastric carcinomas. These results suggest that, although genetic abnormality of BRG1 might be rare, an increased expression of BRG1 might be associated with the development and progression of gastric carcinoma.
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Carcinoma/genética , Proteínas Nucleares/genética , Neoplasias Gástricas/genética , Fatores de Transcrição/genética , Carcinoma/metabolismo , Carcinoma/patologia , DNA Helicases , Análise Mutacional de DNA , Primers do DNA/química , DNA de Neoplasias/análise , Progressão da Doença , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Proteínas Nucleares/metabolismo , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/metabolismo , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Hypermethylation of CpG islands in the promoter region is associated with the silencing of a variety of tumor suppressor genes. DNA repair genes human Mut L homologue 1 (hMLH1) and O(6)-methylguanine-DNA methyltransferase (MGMT) have been shown to be hypermethylated in certain carcinomas. We studied DNA methylation of CpG islands in hMLH1 and MGMT in 50 gastric carcinomas and 10 intestinal metaplastic mucosa samples. We analyzed the methylation status of hMLH1 and MGMT using methylation-specific polymerase chain reaction and DNA sequencing analysis. We measured protein levels of hMLH1 using Western blot and immunohistochemical analysis. CpG island hypermethylation of hMLH1 and MGMT was detected in 11 (22%) and 8 (16%) of the 50 gastric tumors, respectively. Hypermethylation of the promoter was more common in intestinal-type gastric carcinomas than in poorly diffuse-type gastric carcinomas (p = 0.016 and 0.021, respectively; Fisher's exact test). However, hMLH1 promoter hypermethylation did not coincide with MGMT promoter hypermethylation except in 1 patient. Hypermethylation of the hMLH1 promoter but not the MGMT promoter occurred in intestinal metaplastic mucosae. Immunohistochemical analysis revealed a corresponding reduction in hMLH1 protein expression in some of the intestinal metaplastic mucosae. Our results suggest that at least two types of promoter methylation participate in the development of gastric carcinoma. Tumor-specific promoter hypermethylation of hMLH1 may be an early event in carcinogenesis in the stomach.